Histidine residues 139, 363 and 500 are essential for catalytic activity of cofactor-independent phosphoglyceromutase from developing endosperm of the castor plant

Abstract

Cofactor-independent phosphoglyceromutase (PGM) from castor is inactivated by diethyl pyrocarbonate, implicating histidine residues in the catalytic mechanism. Treatment of the inhibited enzyme with 1 M hydroxylamine at pH 7.0 restores the enzyme activity. Spectroscopic data indicate that the inactivation of PGM with diethyl pyrocarbonate is the result of formation of carbethoxyhistidine derivatives. The substrate, 3-phosphoglycerate, substantially protects the enzyme against diethyl pyrocarbonate inactivation, indicating that the histidine residues important in catalysis are at or near the active site of the enzyme. There are 12 conserved histidine residues in all plant PGMs that have been sequenced. In the castor PGM, these conserved histidine residues were changed to either valine (H12V) or alanine (H41A, H65A, H84A, H127A, H139A, H163A, H363A H433A, H471A, H500A and H540A) by in vitro mutagenesis. Expression of these mutant proteins in Escherichia coli produced seven soluble mutant proteins (mutations H41A, H65A, H84A, H139A, H363A, H500A and H540A) and five insoluble mutant proteins (mutations H12V, H127A, H163A, H433A and H471A). Among the seven soluble proteins, four possessed normal PGM activity (mutations H41A, H65A, H84A and H540A) and three (mutations H139A, H363A and H500A) had no catalytic activity. Along with the in-vitro-expressed wild-type enzyme, mutant enzymes [H139A]PGM, [H363A]PGM and [H500A]PGM were purified to homogeneity. Purified wild-type PGM expressed in E. coli was active and had a Km value very close to that of the enzyme purified from castor endosperm, while the three mutant enzymes remained inactive throughout purification. Therefore, histidine residues 139, 363 and 500 appear to be essential for the catalytic activity of the cofactor-independent enzyme, and may be located at the active site. Hence, although the cofactor-dependent and cofactor-independent PGMs have no homology in their primary amino acid sequences, both enzymes appear to utilize histidine residues to mediate the transfers of proton and phospho groups in the reaction, and thus may be functionally and mechanistically convergent.